The present invention, in some embodiments thereof, relates to recombinant protein production in heterologous systems.
Advances in genetic engineering have made possible the production of therapeutics and vaccines for human and animals in the form of recombinant proteins. These biotechnology-derived recombinant proteins form a new class of drugs for many ailments like genetic disorders, cancer, hypertension and AIDS for which there is no better treatment or cure. Unlike chemical drugs, biologicals are the body's own molecules and hence more compatible with biological systems. At present there are more than 100 biotechnology-derived therapeutics and vaccines approved by US FDA for medical use and over 1000 additional drugs and vaccines are in various phases of clinical trials. In addition, use of DNA, proteins and enzymes in diagnostics is increasing exponentially. Industrial uses of enzymes in food, textile, leather, detergent, medicinal chemistry sectors are also increasing rapidly.
The growing need of therapeutic and other applications of enzymes and proteins is presently met by heterologous synthesis of recombinant proteins.
Commonly used expression systems for heterologous protein production include E. coli, baculovirus, yeast, Chinese Hamster Ovary cells (CHO) and plants.
Efficiency of expression of recombinant proteins in heterologous systems depends on many factors, both on the transcriptional level and the translational level.
mRNA translation is controlled at multiple stages and by a diversity of mechanisms. A major part of the control is on the stage of initiation, where ribosomes are recruited and assembled on the mRNA, typically on the 5′ un-translated region (UTR) (1). The elongation phase is governed by the mRNA secondary structure (2), and by the extent of adaptation of the coding sequence to the cellular tRNA pool (3, 4). The abundance of tRNAs that correspond to the different codons in a gene was suggested to determine the speed (5, 6) and accuracy (7) of translation. Thus, codons that are recognized by abundant or rare tRNAs will be respectively referred to here as codons with high and low efficiency (or as codons that are respectively highly or lowly adapted to the tRNA pool). It may be hypothesized that ribosomes will spend less time on high efficiency codons, explore less mismatched tRNAs and thus waste less GTP molecules when translating them, will be less likely to introduce a translation error on such codons and in addition may have lower probability of pre-mature drop off when translating them.
Indeed transcripts whose codons are biased towards the more abundant tRNAs were found to be more highly expressed (5, 8). In addition protein expression levels can be artificially increased by designed mutations that increased their codon-tRNA adaptation (9-12), pointing to a causal relationship between codon usage and expression level. Accordingly, the extent of adaptation between genes to the tRNA pool in different species was found to vary in evolution according to organisms' life style needs (5, 13).